Abstract: This longstanding competing renewal application CA74305 concerns our plans to apply chemical and biochemical approaches in cell signaling to enhance our understanding of protein post-translational modifications (PTMs) and the pathways involved. In the next cycle, we plan to investigate the regulation of three key enzymes, PTEN, WWP2, and AKT1 that are connected to the cancer promoting phospholipid, PIP3 (phosphatidyl 3,4,5-triphosphate). PTEN is one of the most commonly mutated tumor suppressor genes and catalyzes the conversion of PIP3 to PIP2 and in this way antagonizes the action of the PI3-kinase oncogenic signaling pathway. WWP2 is a HECT domain E3 ligase (ubiquitin transferase) that targets PTEN for destruction and is considered to be an oncogene. AKT1, also an oncogene, is a protein Ser/Thr kinase that is activated by PIP3 and drives tumor formation and is an intensively studied cancer therapeutic target. PTEN, WWP2, and AKT1 are each subjected to multiple phosphorylations that are proposed to have important regulatory implications. Our research program will continue to adapt and employ protein semisynthetic methods including the newly described enzyme-catalyzed expressed protein ligation (EPL) to generate these signaling enzymes containing the requisite PTMs and appropriate mimics to interrogate how these phosphorylations influence the structure and function of these key cancer-related enzymes. Our three specific aims are: 1. Optimize enzyme-catalyzed expressed protein ligation (EPL) for protein semisynthesis and apply to the study of PTEN regulation; 2. Elucidate the molecular basis of WWP2 E3 ubiquitin ligase regulation; 3. Determine the role of C-terminal phosphorylation of AKT1 on its structure, activity, mechanism of regulation, substrate specificity, and inhibitor sensitivity. With site-specifically modified signaling enzymes in hand, we will integrate kinetic analysis, X-ray crystallography, protein microarrays, and cell-based studies to clarify key regulatory and signaling features. Upon completion of this research effort, we should have a much clearer understanding of the PIP3 signaling network. Moreover, these studies should pave the way for new therapeutic strategies to combat PTEN-AKT1 pathway dysregulation in cancer.